Assay methods using nicking endonucleases

ABSTRACT

Assay methods and apparatus for the analysis of biopolymers are disclosed. The assays employ nicking endonucleases to enable the generation of flaps on target biomolecules which are detected in nanopore or fluidic channel devices. Identification of flap locations enables a map of the target biomolecule to be derived.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.12/891,343 filed Sep. 27, 2010, which is hereby incorporated byreference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 8, 2010, isnamed NAB-011.txt and is 2,432 bytes in size.

FIELD OF INVENTION

The present invention relates generally to assay methods and devices forthe analysis of biopolymers. Mapping of such biopolymers is contemplatedherein.

BACKGROUND

Identifying the composition and sequence of various biomolecules, suchas human DNA, with accuracy and specificity is of great interest.Mapping and sequencing technology, however, is time consuming andexpensive to develop and implement. For example, sequencing the DNA of asingle individual for the Human Genome Project required over $3 billionof funding.

It is estimated that each person's DNA varies from one another byapproximately 1 base in 1000. Knowledge of such genetic variations amonghuman populations may allow the scientific community to identify genetictrends that are related to various medical predispositions, conditions,or diseases, and may lead to the realization of truly personalizedmedicine where treatments are customized for a given individual based onthat individual's DNA. A reduction in the time and cost of DNA mappingand sequencing is needed to develop such knowledge and to tailor medicaldiagnostics and treatments based on the genetic makeup of individualpatients.

New DNA sequencing technologies produce many short reads (lengths ofsequenced DNA) that are then used to assemble the sequence of the entiresample. These “short-read” technologies have sequenced read lengths offrom 25 bases to 400 bases. For genomes of modest size or complexity,these short reads are incapable of correctly assembling the sequence ofthe sample because of the appearance of repeats in the sequence. DNAmapping may be used to guide the assembly. For instance, restrictionmaps may be used to aid in the assembly of short-read data. More rapidand higher density maps would be useful to enable short-readtechnologies to assemble data.

Hybridization Assisted Nanopore Sequencing (HANS) is a nanopore-basedmethod for sequencing genomic lengths of DNA and other biomolecules. Themethod relies on detecting the position of hybridization of probes onspecific portions of the biomolecule to be sequenced or characterized.

In this method, two reservoirs of solution are separated by ananometer-sized hole, or nanopore, that serves as a fluidic constrictionof known dimensions. The application of a constant DC voltage betweenthe two reservoirs results in a baseline ionic current that is measured.If an analyte is introduced into a reservoir, it may pass through thefluidic channel and change the observed current, due to a difference inconductivity between the electrolyte solution and analyte. The magnitudeof the change in current depends on the volume of electrolyte displacedby the analyte while it is in the fluidic channel. The duration of thecurrent change is related to the amount of time that the analyte takesto pass through the nanopore constriction. The current signals are usedin determining the position of the probes on the biomolecule.Measurement of the probe positions allows for accurate reconstruction ofthe biomolecule sequence using computer algorithms. A need exists forefficient methods and devices capable of rapid and accurate nucleic acidmapping and sequencing for de novo assembly of human genomes. It isdesirable to have long read lengths and to use as little nucleic acidtemplate as possible.

Mapping and sequencing may also be achieved using fluidic nano-channeland micro-channel based devices. In these systems, the analyte is causedto transit through a nano- or micro-channel, and its passage is detectedby electrodes positioned along the length of the channel. In oneembodiment, described in co-pending U.S. patent application Ser. No.12/789,817, filed May 28, 2010, and the teachings of which areincorporated herein by reference in its entirety, a first pair ofelectrodes is positioned longitudinally along the channel to provide areference voltage between them. A second pair of electrodes ispositioned across the channel to define a detection volume. As ananalyte passes through the detection volume, it causes a detectablechange in an electrical property, for example, a change in the referencevoltage. Probes that are hybridized to the analyte cause a furtherchange in the electrical property when they enter the detection volume.Thus, it is possible to detect when the analyte is present in thedetection volume, as well as the absolute or relative position ofhybridized probes as they enter the detection volume.

Despite the foregoing, there remains a need for improved methods anddevices for the analysis of biopolymers, including improved assaymethods for mapping and sequencing such biopolymers.

SUMMARY

The embodiments of the invention relate to assay methods for preparingtarget analyte samples suitable for mapping using nanopore,micro-channel or nano-channel analysis devices.

In one embodiment, the method includes the steps of a) providing adouble-stranded DNA template having first and second DNA strands, eachstrand having a 5′ end and a 3′ end, b) contacting the double-strandedDNA template with a nicking endonuclease to form a nick at asequence-specific nicking location on the first DNA strand, c)conducting a base extension reaction on the first DNA strand along thecorresponding region of the second DNA strand, the reaction starting atthe nick and progressing toward the 3′ end of the first DNA strand tothereby form a single-stranded flap on the template adjacent to thenicking location, and d) coating the double-stranded DNA template with abinding moiety that enhances electrical detection of the template andthe single-stranded flap, to thereby prepare the target analyte.

In another embodiment, the method includes the steps of a) providing adouble-stranded DNA template having first and second DNA strands, eachstrand having a 5′ end and a 3′ end, b) contacting the template with anicking endonuclease to form nicks at sequence-specific locations on thefirst DNA strand, c) conducting a first base extension reaction on thefirst DNA strand along the corresponding region of the second DNAstrand, the reaction starting at each nick and progressing toward the 3′end of the first DNA strand to thereby form single-stranded flap regionson the double-stranded DNA template adjacent to the sequence specificnicking locations, and d) conducting a second base extension reaction onat least one flap region to form at least one double-stranded flap, tothereby prepare the target analyte. In this embodiment, all or a portionof the resulting analyte may be coated with a binding moiety. Thus, inthis embodiment, an additional step of coating the double-stranded flap,as well as the double-stranded DNA template, with a binding moiety maybe employed.

In still another embodiment, the method includes the steps of a)providing a double-stranded DNA template having a first and a second DNAstrand, each DNA strand having a 5′ end and a 3′ end, b) contacting thedouble-stranded DNA template with a nicking endonuclease to form a nickat a sequence-specific nicking location on the first DNA strand, c)conducting a base extension reaction on the first DNA strand along acorresponding region of the second DNA strand, the reaction starting atthe nick and progressing toward the 3′ end of the first DNA strand tothereby form a single-stranded flap on the double-stranded DNA templateadjacent to the sequence-specific nicking location, and d) coating thesingle-stranded flap with a binding moiety that selectively binds withsingle-stranded DNA to enhance electrical detection of thesingle-stranded flap, to thereby prepare the target analyte.

In another embodiment, the method includes the steps of a) providing adouble-stranded DNA template having first and second DNA strands, eachstrand having a 5′ end and a 3′ end, b) contacting the template with anicking endonuclease to form nicks at sequence-specific locations on thefirst DNA strand, c) conducting a first base extension reaction on thefirst DNA strand along the corresponding region of the second DNAstrand, the reaction starting at each nick and progressing toward the 3′end of the first DNA strand to thereby form single-stranded flap regionson the double-stranded DNA template adjacent to the sequence-specificnicking locations, d) conducting a second base extension reaction on atleast one single-stranded flap region to form at least onedouble-stranded flap, e) adding a single-stranded extension to thedouble-stranded flap, and f) hybridizing one or more probes to thesingle-stranded extension, to thereby prepare the target analyte. Thesingle stranded extension may be at least 100 bases in length, and maycomprise a polyT or polyA polymers. The extension may be added to thedouble-stranded flap using a terminal transferase. The probes may betagged with gold particles and may comprise polyA oligomers (in the caseof a polyT extension), or polyT oligomers (in the case of a polyAextension).

In each embodiment above, the biomolecule to be analyzed may bedouble-stranded DNA. The nicking endonucleases may be Nb.BbvCI, Nb.BsmI,NbBsrDI, Nb.BtsI, Nt.AlwI, Nt.BbvCI, Nt.BsmAI, Nt.BspQI, Nt.BstNBI, andNt.CviPII, used either alone or in various combinations; however, anynicking endonuclease may be employed, either alone or in combination.Base extension reactions may be achieved by contacting a DNA strand witha polymerase, one or more nucleotides, a ligase, or any combinationthereof. The binding moiety may be a protein. Examples of suitableproteins include RecA, T4 gene 32 protein, f1 geneV protein, humanreplication protein A, Pf3 single-stranded binding protein, adenovirusDNA binding protein, and E. coli single-stranded binding protein.

Further embodiments of the invention relate to target analytescomprising double-stranded DNA fragments having one or moredouble-stranded DNA flaps, and target analytes comprisingdouble-stranded DNA fragments having one or more single- ordouble-stranded DNA flaps that have been coated with a binding moiety.DNA fragments having one or more double-stranded flaps withsingle-stranded extensions are contemplated as well. The extensions maybe hybridized with oligonucleotide probes, and the probes may includetags such as gold particles.

The target analytes prepared by the methods of the present invention areconfigured for the detection of positional information in a nanoporesystem, as well as in a fluidic channel system employing nano-channelsor micro-channels. As such, embodiments of the invention relate tomonitoring changes in an electrical property across a nanopore or afluidic channel as the target analytes made using the methods describedabove are translocated through the nanopore or fluidic channel. Themonitored changes are indicative of double-stranded regions of thetarget analyte as well as flap regions. Further, embodiments of theinvention relate to using the detected changes in the electricalproperty to differentiate between double-stranded and flap regions onthe target analyte. The differentiation may be used to determine nicklocations and thereby map at least a portion of the DNA template.

Thus, in one embodiment, the present invention relates to the method ofa) providing a double-stranded DNA template having first and second DNAstrands, each strand having a 5′ end and a 3′ end, b) contacting thedouble-stranded DNA template with a nicking endonuclease to form a nickat a sequence-specific nicking location on the first DNA strand, c)conducting a base extension reaction on the first DNA strand along thecorresponding region of the second DNA strand, the reaction starting atthe nick and progressing toward the 3′ end of the first DNA strand tothereby form a single-stranded flap on the template adjacent to thenicking location, d) coating the double-stranded DNA template with abinding moiety that enhances electrical detection of the template andthe single-stranded flap, to thereby prepare the target analyte, and e)monitoring changes in an electrical property as the target analyte istranslocated through a nanopore or across a fluidic micro- ornano-channel, where the changes in the electrical property areindicative of double-stranded regions of the target analyte and of thesingle-stranded flap regions. The additional step of differentiatingbetween the double-stranded template and single-stranded flap regions,based at least in part, on detected changes in the electrical property,to determine nick locations and map at least a portion of thedouble-stranded DNA template may be employed as well.

In another embodiment, the present invention relates to the method of a)providing a double-stranded DNA template having first and second DNAstrands, each strand having a 5′ end and a 3′ end, b) contacting thetemplate with a nicking endonuclease to form nicks at sequence-specificlocations on the first DNA strand, c) conducting a first base extensionreaction on the first DNA strand along the corresponding region of thesecond DNA strand, the reaction starting at each nick and progressingtoward the 3′ end of the first DNA strand to thereby formsingle-stranded flap regions on the double-stranded DNA templateadjacent to the sequence specific nicking locations, d) conducting asecond base extension reaction on at least one flap region to form atleast one double-stranded flap, to thereby prepare the target analyte,and e) monitoring changes in an electrical property as the targetanalyte is translocated through a nanopore or across a fluidic micro- ornano-channel, where the changes in the electrical property areindicative of double-stranded regions of the target analyte and of thedouble-stranded flap regions. The additional step of differentiatingbetween the double-stranded template and double-stranded flap regions,based at least in part, on detected changes in the electrical property,to determine nick locations and map at least a portion of thedouble-stranded DNA template may be employed as well.

In still another embodiment, the method includes the steps of a)providing a double-stranded DNA template having a first and a second DNAstrand, each DNA strand having a 5′ end and a 3′ end, b) contacting thedouble-stranded DNA template with a nicking endonuclease to form a nickat a sequence-specific nicking location on the first DNA strand, c)conducting a base extension reaction on the first DNA strand along acorresponding region of the second DNA strand, the reaction starting atthe nick and progressing toward the 3′ end of the first DNA strand tothereby form a single-stranded flap on the double-stranded DNA templateadjacent to the sequence-specific nicking location, d) coating thesingle-stranded flap with a binding moiety that selectively binds withsingle-stranded DNA to enhance electrical detection of thesingle-stranded flap, to thereby prepare the target analyte, and e)monitoring changes in an electrical property as the target analyte istranslocated through a nanopore or across a fluidic micro- ornano-channel, where the changes in the electrical property areindicative of double-stranded regions of the target analyte and of thesingle-stranded flap regions. The additional step of differentiatingbetween the double-stranded template and single-stranded flap regions,based at least in part, on detected changes in the electrical property,to determine nick locations and map at least a portion of thedouble-stranded DNA template may be employed as well.

In another embodiment, the method includes the steps of a) providing adouble-stranded DNA template having first and second DNA strands, eachstrand having a 5′ end and a 3′ end, b) contacting the template with anicking endonuclease to form nicks at sequence-specific locations on thefirst DNA strand, c) conducting a first base extension reaction on thefirst DNA strand along the corresponding region of the second DNAstrand, the reaction starting at each nick and progressing toward the 3′end of the first DNA strand to thereby form single-stranded flap regionson the double-stranded DNA template adjacent to the sequence-specificnicking locations, d) conducting a second base extension reaction on atleast one single-stranded flap region to form at least onedouble-stranded flap, e) adding a single-stranded extension to thedouble-stranded flap, and f) hybridizing one or more probes to thesingle-stranded extension, to thereby prepare the target analyte.Changes in an electrical property may be monitored as the target analyteis translocated through a nanopore or across a fluidic micro- ornano-channel, where the changes in the electrical property areindicative of double-stranded regions of the target analyte and of theextended flap regions. The additional step of differentiating betweenthe double-stranded template and extended flap regions, based at leastin part, on detected changes in the electrical property, to determinenick locations and map at least a portion of the double-stranded DNAtemplate may be employed as well.

In another embodiment, the method includes the steps of a) providing adouble-stranded DNA template having first and second DNA strands, eachstrand having a 5′ end and a 3′ end, b) contacting the double-strandedDNA template with a nicking endonuclease to form a nick at asequence-specific nicking location on the first DNA strand, c)conducting a base extension reaction on the first DNA strand along thecorresponding region of the second DNA strand, the reaction starting atthe nick and progressing toward the 3′ end of the first DNA strand tothereby form a single-stranded flap on the template adjacent to thenicking location, and d) monitoring changes in an electrical property asthe target analyte is translocated through a nanopore or across afluidic micro- or nano-channel, where the changes in the electricalproperty are indicative of double-stranded regions of the target analyteand of the single-stranded flap regions. The additional step ofdifferentiating between the double-stranded template and single-strandedflap regions, based at least in part, on detected changes in theelectrical property, to determine nick locations and map at least aportion of the double-stranded DNA template may be employed as well.

As before, in each embodiment above, the biomolecule to be analyzed maybe double-stranded DNA. The nicking endonucleases may be Nb.BbvCI,Nb.BsmI, NbBsrDI, Nb.BtsI, Nt.AlwI, Nt.BbvCI, Nt.BsmAI, Nt.BspQI,Nt.BstNBI, and Nt.CviPII, used either alone or in various combinations;however, any nicking endonuclease may be employed, either alone or incombination. Base extension reactions may be achieved by contacting aDNA strand with a polymerase, one or more nucleotides, a ligase, or anycombination thereof. If used, the binding moiety may be a protein.Examples of suitable proteins include RecA, T4 gene 32 protein, f1 geneVprotein, human replication protein A, Pf3 single-stranded bindingprotein, adenovirus DNA binding protein, and E. coli single-strandedbinding protein.

In cases where a single-stranded extension is formed on adouble-stranded flap region, the single stranded extension may be atleast 100 bases in length, and may comprise a polyT or polyA polymers.The extension may be added to the double-stranded flap using a terminaltransferase. The probes may be tagged, for example, with gold particlesand may comprise polyA oligomers (in the case of a polyT extension), orpolyT oligomers (in the case of a polyA extension).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic depiction of a DNA molecule (SEQ ID NOS 5-6,respectively, in order of appearance).

FIG. 2 a is a schematic depiction of a DNA molecule (SEQ ID NOS 5-6,respectively, in order of appearance) showing the recognition andnicking site for the nicking endonuclease Nt.BbvCI.

FIG. 2 b is a schematic depiction of a DNA molecule (bottom strand isdisclosed as SEQ ID NO: 6) subsequent to reaction with the nickingendonuclease Nt.BbvCI.

FIG. 3 is a schematic depiction of a nicked DNA molecule (SEQ ID NOS 7and 6, respectively, in order of appearance) subsequent to a baseextension reaction beginning at the nicking site.

FIG. 4 a is a schematic depiction of the DNA molecule of FIG. 3subsequent to contact of the molecule with a binding moiety that bindsto single- and double-stranded DNA.

FIG. 4 b is a schematic depiction of the DNA molecule of FIG. 3subsequent to contact of the molecule with a binding moiety thatpreferentially binds to single-stranded DNA.

FIG. 5 a is a schematic depiction of a DNA molecule showing arecognition and nicking site for a nicking endonuclease.

FIG. 5 b is a schematic depiction of a DNA molecule subsequent toreaction with a nicking endonuclease.

FIG. 5 c is a schematic depiction of a nicked DNA molecule subsequent toa first base extension reaction to form a flap.

FIG. 5 d is a schematic depiction of a DNA molecule subsequent tohybridization of an oligonucleotide probe to the flap.

FIG. 5 e is a schematic depiction of a DNA molecule subsequent to asecond base extension reaction to form a double-stranded flap.

FIG. 5 f is a schematic depiction of a DNA molecule having adouble-stranded flap.

FIG. 6 is a schematic depiction of the DNA molecule of FIG. 5 fsubsequent to contact of the molecule with a binding moiety.

FIG. 7 a is a schematic depiction of a DNA molecule having a pluralityof closely-spaced recognition and nicking sites subsequent to reactionwith a nicking endonuclease.

FIG. 7 b is a schematic depiction of the DNA molecule of FIG. 7 asubsequent to a first base extension reaction to form a plurality offlaps.

FIG. 7 c is a schematic depiction a portion of the DNA molecule of FIG.7 b depicting an isolation of a single flap.

FIG. 7 d is a schematic depiction of the portion of the DNA molecule ofFIG. 7 c subsequent to hybridization of the flap with a probe and use ofa base extension reaction to convert the flap into double-stranded DNA.

FIG. 7 e is a schematic depiction of the portion of the DNA molecule(SEQ ID NO: 8) of FIG. 7 d subsequent to the addition of asingle-stranded extension to the flap.

FIG. 7 f is a schematic depiction of the portion of the DNA molecule(SEQ ID NO: 8) of FIG. 7 e subsequent to hybridization of the extensionwith complementary oligonucleotide probes.

FIG. 7 g is a schematic depiction of the portion of the DNA molecule(SEQ ID NO: 8) of FIG. 7 e subsequent to hybridization of the extensionwith tagged complementary oligonucleotide probes.

FIG. 8 a is a schematic depiction of a DNA molecule having a flap in ananopore apparatus.

FIG. 8 b is a schematic depiction of a current measurement waveform as aDNA molecule having a flap translocates through the nanopore apparatusof FIG. 8 a.

FIG. 9 is a schematic depiction of a fluidic channel apparatus usefulfor mapping the analytes of the present invention, in which the fluidicchannel may be a nano-channel or a micro-channel.

FIG. 10 a is a schematic depiction of an electrical potentialmeasurement as a DNA molecule having a flap enters a detection volume inthe apparatus of FIG. 9.

FIG. 10 b is a schematic depiction of an electrical potentialmeasurement as a flap on a DNA molecule enters a detection volume in theapparatus of FIG. 9.

FIG. 10 c is a schematic depiction of an electrical potentialmeasurement as a flap on a DNA molecule exits a detection volume in theapparatus of FIG. 9.

FIG. 10 d is a schematic depiction of an electrical potentialmeasurement as a DNA molecule having a flap exits a detection volume inthe apparatus of FIG. 9.

FIG. 11 is a schematic depiction of a nano-channel or micro-channelapparatus having multiple detection volumes.

DETAILED DESCRIPTION

As used in this description and the accompanying claims, the followingterms shall have the meanings given, unless the context indicatesotherwise:

An “analyte” or “target” means a double-stranded DNA biomolecule.

A “DNA template” means a DNA molecule that serves as a pattern for thesynthesis of an additional molecular structure.

A “binding moiety” means an entity, such as a protein, capable ofproviding a coating on an analyte.

An “endonuclease” is an enzyme that cleaves the phosphodiester bondwithin a polynucleotide chain, in contrast with an exonuclease, whichcleaves phosphodiester bonds at the end of a polynucleotide chain.

A “restriction endonuclease” or “restriction enzyme” cleaves DNA atspecific sites.

A “nicking endonuclease” or “nickase” is a form of restrictionendonuclease. Restriction endonucleases recognize specific nucleotidesequences in double-stranded DNA and generally cleave both strands. Somesequence-specific endonucleases, however, cleave only one of thestrands. These endonucleases are known as nicking endonucleases.

The translocation of biomolecules and biomolecule/protein complexesthrough a nanopore, a nano-channel or a micro-channel sequencing systemmay include detecting an electrical signal indicative of the passage ofthe biomolecule through the system, as well as an electrical signalindicative of the passage of single- or double-stranded flaps formed onthe biomolecule through the system. The biomolecule and/or the flaps maybe coated with a binding moiety such as a protein. The time fortranslocation may be indicative of the length of the biomolecule. Thedetection step may discriminate between coated, uncoated, or multiplycoated regions, as a coated region may have a signal about ten timesthat of an uncoated region. Increasing the signal-to-noise ratio mayincrease confidence in the detection of flaps formed on the biomolecule.Positional information of flaps on the target biomolecule may be used toidentify nicking sites, and thereby facilitates the mapping of thebiomolecule.

Referring to FIG. 1, a DNA molecule 10, e.g., a double-stranded DNAtemplate, is schematically depicted. The molecule 10 comprises two DNAstrands, e.g., first and second strands 12, 14 positioned inanti-parallel relation to one another. The two DNA strands may also bereferred to as top and bottom strands 12, 14. Each of the two opposingstrands 12, 14 may be sequentially formed from repeating groups ofnucleotides 16 where each nucleotide 16 consists of a phosphate group,2-deoxyribose sugar and one of four nitrogen-containing bases. Thenitrogen-containing bases include cytosine (C), adenine (A), guanine (G)and thymine (T). Each DNA strand has a 5′ end and a 3′ end. Inparticular, the DNA strands 12, 14 are read in a particular direction,from the top (called the 5′ or “five prime” end) to the bottom (calledthe 3′ or “three prime” end). Similarly, RNA molecules arepolynucleotide chains, which differ from those of DNA by having ribosesugar instead of deoxyribose and uracil bases (U) instead of thyminebases (T).

The bases in the molecules do not share an equal affinity for oneanother. Thymine (T) bases favor binding with adenine (A) bases, whilecytosine (C) bases favor binding with guanine (G) bases. Binding ismediated via hydrogen bonds that exist between the opposing base pairs.For example, base A binds to base T using two hydrogen bonds, while baseC binds to base G using three hydrogen bonds.

Nicking endonucleases useful in embodiments of the present inventioninclude Nb.BbvCI, Nb.BsmI, NbBsrDI, Nb.BtsI, Nt.AlwI, Nt.BbvCI,Nt.BsmAI, Nt.BspQI, Nt.BstNBI, and Nt.CviPII, used either alone or invarious combinations. As noted above, nickases are sequence-specificendonucleases which are characterized in that they cleave only onestrand of double-stranded DNA at the recognition site.

The nickase Nb.BbvCI is derived from an E. coli strain expressing analtered form of the BbvCI restriction genes [Ra+:Rb(E177G)] fromBacillus brevis. It nicks at the following recognition site (with“{hacek over ( )}” specifying the nicking site and “N” representing anyone of C, A, G or T):

5′ . . . C C T C A G C . . . 3′ 3′ . . . G G A G T{hacek over( )}C G . . . 5′

The nickase Nb.BsmI is derived from an E. coli strain that carries thecloned BsmI gene from Bacillus stearothermophilus NUB 36. It nicks atthe following recognition site:

5′ . . . G A A T G C N . . . 3′ 3′ . . . C T T A C{hacek over( )}G N . . . 5′

The nickase Nb.BsrDI is derived from an E. coli strain expressing onlythe large subunit of the BsrDI restriction gene from Bacillusstearothermophilus D70. It nicks at the following recognition site:

5′ . . . G C A A T G N N . . . 3′ 3′ . . . C G T T A C{hacek over( )}N N . . . 5′

The nickase Nb.BtsI is derived from an E. coli strain expressing onlythe large subunit of the BtsI restriction gene from Bacillusthermoglucosidasius. It nicks at the following recognition site:

5′ . . . G C A G T G N N . . . 3′ 3′ . . . C G T C A C{hacek over( )}N N . . . 5′

The nickase Nt.AlwI is an engineered derivative of AlwI which catalyzesa single-strand break four bases beyond the 3′ end of the recognitionsequence on the top strand. It is derived from an E. coli straincontaining a chimeric gene encoding the DNA recognition domain of AlwIand the cleavage/dimerization domain of Nt.BstNBI. It nicks at thefollowing recognition site:

(SEQ ID NO: 1) 5′ . . . G G A T C N N N N{hacek over ( )}N . . . 3′(SEQ ID NO: 2) 3′ . . . C C T A G N N N N N . . . 5′

The nickase Nt.BbvCI is derived from an E. coli strain expressing analtered form of the BbvCI restriction genes [Ra(K169E):Rb+] fromBacillus brevis. It nicks at the following recognition site:

5′ . . . C C{hacek over ( )}T C A G C . . . 3′ 3′. . . G G A G T C G . . . 5′

The nickase Nt.BsmAI is derived from an E. coli strain expressing analtered form of the BsmAI restriction genes from Bacillusstearothermophilus A664. It nicks at the following recognition site:

5′ . . . G T C T C N{hacek over ( )}N . . . 3′ 3′. . . C A G A G N N . . . 5′

The nickase Nt.BspQI is derived from an E. coli strain expressing anengineered BspQI variant from BspQI restriction enzyme. It nicks at thefollowing recognition site:

5′ . . . G C T C T T C N{hacek over ( )} . . . 3′ 3′. . . C G A G A A G N . . . 5′

The nickase Nt.BstNBI catalyzes a single strand break four bases beyondthe 3′ side of the recognition sequence. It is derived from an E. colistrain that carries the cloned Nt.BstNBI gene from Bacillusstereothermophilus 33M. It nicks at the following recognition site:

(SEQ ID NO: 3) 5′ . . . G A G T C N N N N{hacek over ( )}N . . . 3′(SEQ ID NO: 4) 3′ . . . C T C A G N N N N N . . . 5′

The nickase Nt.CviPII cleaves one strand of DNA of a double-stranded DNAsubstrate. The final product on pUC19 (a plasmid cloning vector) is anarray of bands from 25 to 200 base pairs. CCT is cut less efficientlythan CCG and CCA, and some of the CCT sites remain uncleaved. It isderived from an E. coli strain that expresses a fusion of Mxe GyrAintein, chitin-binding domain and a truncated form of the Nt.CviPIInicking endonuclease gene from Chlorella virus NYs-1. It nicks at thefollowing recognition site:

5′ . . . {hacek over ( )}C C D . . . 3′ 3′ . . . G G H . . . 5′

Each of the restriction endonucleases described above is available fromNew England Biolabs of Ipswich, Mass.

It should be understood that the invention is not intended to be limitedto the nicking endonucleases described above; rather, it is anticipatedthat any endonuclease capable of providing a nick in a double-strandedDNA molecule may be used in accordance with the methods of the presentinvention.

One embodiment of the present invention is depicted in FIGS. 2 a-4 a or4 b. In FIG. 2 a, the DNA molecule 10 of FIG. 1 is shown. Therecognition sequence of the nicking endonuclease Nt.BbvCI is indicatedby the boxed area 18, and the specific nicking site 20 of that nickingendonuclease is identified. The double-stranded DNA template iscontacted with the nicking endonuclease. In FIG. 2 b, the nickingendonuclease Nt.BbvCI has cleaved the top strand of the DNA moleculeleaving a nick 20′ at a sequence-specific nicking location on the topDNA strand 12.

As shown in FIG. 3, a base extension reaction 22, such as a primerextension reaction, utilizing for example, a polymerase, one or morenucleotides, a ligase, or any combination thereof is carried outbeginning at the nick site (indicated by a dot). The base extensionreaction is conducted on the top DNA strand along (and pursuant to) thecorresponding region of the bottom DNA strand. In such reactions, thatform a nucleic acid complementary to a nucleic acid template, a primercomplementary to a single-stranded DNA template is typically employed.Starting at the primer, a DNA polymerase may be used to addmononucleotides complementary to the template at the 3′ end of theprimer. Various base extension reactions will be familiar to those ofordinary skill in the art. As the base extension reaction progressestoward the 3′ end of the top strand, a flap 24 of single-stranded DNA isformed from the original top strand of the DNA molecule. The flap 24 issingle-stranded, and is adjacent to the sequence-specific nickinglocation. The base extension reaction is allowed to proceed until theflap 24 has reached a desired length, resulting in the formation of atarget analyte including the double-stranded DNA molecule and a flap.the presence of the flap aids in the detection via, for example, HANS,or other nanopore-, nanochannel-, or microchannel-based detectiontechniques. In one embodiment, it is preferred that the flap 24 be atleast one hundred (100) bases in length, however, the invention is notintended to be limited as such.

Finally, as shown in FIGS. 4 a and 4 b, a binding moiety may be used tocoat the DNA molecule or portions thereof. In FIG. 4 a, the bindingmoiety 30 has been used to coat both strands 12, 14 of the DNA moleculeas well as the single-stranded flap 24. Alternatively, as shown in FIG.4 b, a binding moiety 30′ having a preference for single-stranded DNAhas been used to coat only the single-stranded flap formed on the DNAmolecule via the method described above.

The binding moiety 30 may include or consist essentially of a protein orother composition which binds to single- and/or double-stranded DNA. Inone embodiment, the binding moiety is the protein RecA. The bindingmoiety may enhance electrical detection of the double-stranded DNAtemplate and flap via, for example, HANS, or other nanopore-,nanochannel-, or microchannel-based detection techniques.

Protein RecA from E. coli typically binds single- or double-stranded DNAin a cooperative fashion to form filaments containing the DNA in a coreand an external sheath of protein (McEntee, K.; Weinstock, G. M.;Lehman, I. R. Binding of the RecA Protein of Escherichia coli to Single-and Double-Stranded DNA. J. Biol. Chem. 1981, 256, 8835, incorporated byreference herein in its entirety). DNA has a diameter of about 2 nm,while DNA coated with RecA has a diameter of about 10 nm. Thepersistence length of the DNA increases to around 950 nm, in contrast to0.75 nm for single-stranded DNA or 50 nm for double-stranded DNA. T4gene 32 protein is known to cooperatively bind single-stranded DNA(Alberts, B. M.; Frey, L. T4 Bacteriophage Gene32: A Structural Proteinin the Replication and Recombination of DNA. Nature, 1970, 227,1313-1318, incorporated by reference herein in its entirety). E. colisingle-stranded binding protein binds single-stranded DNA in severalforms depending on salt and magnesium concentrations (Lohman, T. M.;Ferrari, M. E. Escherichia Coli Single-Stranded DNA-Binding Protein:Multiple DNA-Binding Modes and Cooperativities. Ann. Rev. Biochem. 1994,63, 527-570, incorporated by reference herein in its entirety). The E.coli single-stranded binding protein may form a varied coating on thebiomolecule. The f1 geneV protein is known to coat single-stranded DNA(Terwilliger, T. C. Gene V Protein Dimerization and Cooperativity ofBinding of poly(dA). Biochemistry 1996, 35, 16652, incorporated byreference herein in its entirety), as is human replication protein A(Kim, C.; Snyder, R. O.; Wold, M. S. Binding properties of replicationprotein A from human and yeast cells. Mol. Cell Biol. 1992, 12, 3050,incorporated by reference herein in its entirety), Pf3 single-strandedbinding protein (Powell, M. D.; Gray, D. M. Characterization of the Pf3single-strand DNA binding protein by circular dichroism spectroscopy.Biochemistry 1993, 32, 12538, incorporated by reference herein in itsentirety), and adenovirus DNA binding protein (Tucker, P. A.;Tsernoglou, D.; Tucker, A. D.; Coenjaerts, F. E. J.; Leenders, H.;Vliet, P. C. Crystal structure of the adenovirus DNA binding proteinreveals a hook-on model for cooperative DNA binding. EMBO J. 1994, 13,2994, incorporated by reference herein in its entirety). Theprotein-coated DNA may then be translocated through a nanopore as hasbeen demonstrated with RecA bound to double-stranded DNA (Smeets, R. M.M.; Kowalczyk, S. W.; Hall, A. R.; Dekker, N. H.; Dekker, C.Translocation of RecA-Coated Double-Stranded DNA through Solid-StateNanopores. Nano Lett. 2009, incorporated by reference herein in itsentirety). The protein coating functions in the same manner forsingle-stranded DNA regions and double-stranded DNA regions.

In another embodiment of the present invention, the flap formed by thebase extension reaction illustrated in FIG. 3 may be converted from asingle-stranded flap to a double-stranded flap. In this embodiment,prior to contacting the target analyte with the binding moiety, thesingle-stranded flap is exposed to a random selection of oligomer probeswith the expectation that at least one will hybridize with a portion ofthe flap and act as a primer. Following such hybridization, a baseextension reaction is carried out to cause the flap to becomedouble-stranded.

More specifically, a double-stranded DNA molecule 10 having first andsecond DNA strands 12, 14 is shown in FIG. 5 a. The DNA molecule 10 hasa recognition sequence that identifies a nicking site 20 for apreselected nicking endonuclease. As shown in FIG. 5 b, upon exposure tothe nicking endonuclease, a nick 20′ is formed on the DNA molecule 10,i.e., on sequence-specific location on the first DNA strand. By using apredetermined nicking endonuclease with a known recognition sequence, ifthe site of the nick can be identified, the location of specificrecognition sequence can be determined, thereby allowing the molecule tobe mapped.

Following nick 20′ formation, a first base extension reaction 22,beginning at the nick site, (indicated by a dot), is carried out. FIG. 5c shows that as the base extension reaction proceeds toward the 3′ endof the top strand, a flap 24 of single-stranded DNA is formed from theoriginal top strand of the DNA molecule. The base extension reaction isallowed to proceed until the flap 24 has reached a desired length. Inone embodiment, it is preferred that the flap 24 be at least one hundred(100) bases in length, however as discussed with respect to FIG. 3, theinvention is not intended to be limited as such.

In FIG. 5 d, the flap 24 is exposed to a random selection ofoligonucleotide probes. The identity of the probes need not be known;rather, it is the expectation that at least one such probe 32 willhybridize to a portion of the flap 24 to act as a primer forming a siteat which a second base extension 34 may be carried out as shown in FIG.5 e. The second base extension can be performed by contacting the flapwith a polymerase, one or more nucleotides, a ligase, or any combinationthereof. The resulting target analyte 50 comprising a double-strandedDNA base 10 with a double-stranded flap 24′ is depicted in FIG. 5 f.

In FIG. 6, a binding moiety 30 has been used to coat both strands 12, 14of the DNA molecule as well as the double-stranded flap 24′. As in theprevious embodiments, the binding moiety 30 may be a protein or othercomposition which binds to single- and double-stranded DNA, such as theprotein RecA, T4 gene 32 protein, f1 geneV protein, human replicationprotein A, Pf3 single-stranded binding protein, adenovirus DNA bindingprotein, and E. coli single-stranded binding protein. The binding moietyenhances electrical detection of the target analyte.

In some instances, the recognition and nicking sites may be spacedrelatively closely together. In these instances, use of the methodsdescribed above may result in small, less readily detectable flaps.Since the flap is formed by the single strand between nicks, if thenicks are closely spaced, flap length is limited. Furthermore, in theseinstances, if the base extension which results in the flap is allowed toproceed too far, the single strand will be excised entirely from theanalyte, resulting in the loss of the flap and thereby the data itprovides. A solution to this problem is depicted in FIGS. 7 a-7 g.

FIG. 7 a depicts a DNA molecule 10 having first 12 and second 14strands. Strand 12 has been reacted with a nicking endonuclease whichcreated closely-spaced nicks 20′. A short segment of single-stranded DNA12′, which will form one flap, is positioned between the nicks.

In FIG. 7 b, a base extension reaction has been carried out to formflaps 24 at each of the nicks. Due to the close spacing between thenicks, the base extension reaction is preferably terminated beforesegment 12′ is excised entirely from the molecule. As a result, a shortflap, which is harder to detect, is formed. Furthermore, since the baseextension is not isolated to the single short flap of segment 12′, butrather, applies to the entire molecule, the early termination of thebase extension results in all flaps being small and less readilydetectable. The boxed area 19 is used to indicate the portion of themolecule containing one flap that is depicted in FIGS. 7 c-7 g.

FIG. 7 c depicts a single short flap 24 (isolation from box 19)following termination of the base extension.

In FIG. 7 d, a double-stranded flap 24′ is depicted. Thisdouble-stranded flap has been formed using the method shown in FIG. 5 e.Specifically, the single-stranded flap has been exposed to a randomselection of oligonucleotide probes, and one such probe hybridized tothe flap and acted as a primer, allowing a second base extension to becarried out. The result is formation of a short, double-stranded flap24′.

FIG. 7 e depicts the molecule of FIG. 7 d following an extension of theflap. Specifically, following the base extension reaction which formedthe double-stranded probe, the molecule is reacted with a terminaltransferase to allow extension of the double-stranded flap. Theextension 40 occurs on the strand having an exposed 3′ end. While anysequence could be extended from the flap 24′, it a preferable to formthe extension as a polymer of a single base, with polyA and polyTextensions preferred. In the embodiment of FIG. 7 e, a polyT extensionis shown, however, the invention is not intended to be limited as such.Regardless of the specific sequence of the extension, it may be anylength desirable for detection, with lengths of several hundred basespreferred.

As a result of the extensions formed on the flaps, detection of theflaps using the methods described below is enhanced as compared to shortflaps lacking such extensions. Further enhancements to flap and moleculedetection are envisioned as well. For example, the molecule may bereacted with a binding moiety using the methods outlined in FIGS. 4 aand 4 b. Alternatively, tagged probes may be hybridized to theextensions. Thus, as shown in FIG. 7 f, oligonucleotide probes havingspecificity to the sequence of the extension may be employed. In FIG. 7f, the polyT extension 40 is hybridized with complementary polyAoligonucleotide probes 42. As shown in FIG. 7 g, these probes 42 may beprovided with tags 44 connected to the probes 42 by a linker 46. Whileany of a wide variety of tags may be employed, in one preferredembodiment, the tags 44 comprise gold beads. It is intended that thetags will enhance detection of the flaps formed at each of the originalnicking sites. Regardless of the enhancement method used, the principleis the same; i.e., detection of the flaps allows determination of therelative position of the original nicking sites. Since the particularnicking endonuclease used to form the nicks is known, determination ofthe identity and relative location of the nicking sites is enabled.

The target analytes described herein may be configured for detection ofpositional information in a nanopore and/or a fluidic channel, i.e., amicro-channel or nano-channel system. Mapping of target analytes may becarried out using electrical detection methods employing nanopores,nano-channels or micro-channels using the methods described in U.S.patent application Ser. No. 12/789,817, filed May 28, 2010, theteachings of which have previously been incorporated herein byreference. It is contemplated that such methods may be applied touncoated analytes having single- or double-stranded flaps, or toanalytes having single- or double-stranded flaps where one or both ofthe base molecule and the single- or double-stranded flap is coated witha binding moiety.

In one embodiment, current across a nanopore is measured duringtranslocation of a DNA strand through the nanopore as shown in FIG. 8 a.When used in embodiments of the present invention, a nanopore may have adiameter selected from a range of about 1 nm to about 1 μm. Morepreferably the nanopore has a diameter that is between about 2.3 nm andabout 100 nm. Even more preferably the nanopore has a diameter that isbetween about 2.3 nm and about 50 nm. Changes in an electrical propertyacross a nanopore may be monitored as the target analyte is translocatedtherethrough, with changes in the electrical property being indicativeof double-stranded regions of the target analyte and of thesingle-stranded or double-stranded flap regions.

Specifically, for nanopore 100, a measurable current produced byelectrodes 120, 122 runs parallel 110 to the movement of the targetanalyte 50, i.e., a DNA molecule having a flap 24. Variations in currentare a result of the relative diameter of the target analyte 50 as itpasses through the nanopore 100. This relative increase in volume of thetarget analyte 50 passing through the nanopore 100 causes a temporaryinterruption or decrease in the current flow through the nanopore,resulting in a measurable current variation. Portions of the targetanalyte 50 including a flap 24 are larger in diameter than portions ofthe target analyte that do not include a flap. As a result, when theflap 24 passes through the nanopore 100, further interruptions ordecreases in the current flow between electrodes 120, 122 occurs. Thesechanges in current flow are depicted in the waveform 200 in FIG. 8 b.

Analysis of the waveform 200 permits differentiation betweendouble-stranded and flap regions of the target analyte based, at leastin part, on the detected changes in the electrical property, to therebydetermine nick locations and map at least a portion of thedouble-stranded DNA template. In FIG. 8 b, the waveform 200 depicts thechanges in a detected electrical property as the target analyte passesthrough the nanopore, and may be interpreted as follows. Currentmeasurement 210 represents measured current prior to passage of the DNAmolecule 10 having a flap formed thereon, i.e., the target analyte,through the nanopore 100 from the cis side to the trans side. As thetarget analyte enters the nanopore 100, from the cis side of thenanopore, the current is partially interrupted forming a first trough220 in the recorded current. Once the flap 24 of the target analyteenters the nanopore 100, a further decrease in current occurs, causing adeeper, second trough 230 in the current measurement. Upon passage ofthe flap 24 entirely through the nanopore 100, a distal portion of thetarget analyte may remain in the nanopore. This causes the measuredcurrent 240 to rise to approximately the level of the first trough 220.Finally, once the entire target analyte has passed completely throughthe nanopore 100 to the trans side, the measured current 250 returns toa level approximating that of the initial level 210. The currentvariation measurements are recorded as a function of time. As a result,the periodic variations in current indicate where, as a function ofrelative or absolute position, the flaps 24 are formed on the targetanalyte 10. Since the flaps are formed at recognition sites for thespecific nicking endonucleases used in flap formation, the relative orabsolute position of the specific sequences associated with therecognition site for the particular nicking endonuclease employed may bedetermined. This allows mapping of those specific sequences on thetarget analyte. Multiple maps produced using multiple nickingendonucleases may be generated.

The use of a binding moiety, such as the protein RecA, may furtherenhance detection of target analytes and flap regions on target analytesbecause the added bulk of the binding moiety coating causes greatercurrent deflections.

In another embodiment, an electrical property such as electricalpotential or current is measured during translocation of a DNA strandthrough a nano-channel or micro-channel as shown in FIGS. 9 through 11.One embodiment of a fluidic channel apparatus is shown schematically inFIG. 9. In FIG. 9, the apparatus 300 comprises a fluidic micro-channelor nano-channel 302. The fluidic channel may be a micro-channel having awidth selected from a range of about 1 μm to about 25 μm or anano-channel having a width selected from a range of about 10 nm toabout 1 μm. In the case of a micro-channel, the depth may be selectedfrom a range of about 200 nm to about 5 μm, whereas in the case of anano-channel, the depth may be selected from a range of about 10 nm toabout 1 μm. In either case, the channel may have a length selected froma range of about 1 μm to about 10 cm.

A first pair of electromotive electrodes 304, 304′ is connected to avoltage source 306 and positioned in a spaced apart relationship in thechannel. When a potential is applied to the electromotive electrodes,these electrodes provide an electrical current along the channel and maybe used to provide or enhance a driving force 308 to a target analyte 50in the channel. Other driving forces such as pressure or chemicalgradients are contemplated as well. A second pair of electrodes 312,312′, i.e., detector electrodes, is positioned preferably substantiallyperpendicular to the channel in a spaced apart relationship to define adetection volume 314. The second pair of detector electrodes 312, 312′is connected to a detector 316, such as a voltmeter, which monitors anelectrical property in the detection volume 314. In an embodiment wherethe detector 316 is a voltmeter, an electrical potential between thepair of detector electrodes 312, 312′, is measured across the detectionvolume 314.

The operation of the device is depicted schematically in FIGS. 10 a-10 din which changes in an electrical property across a fluidic channel aremonitored, as the target analyte is translocated therethrough, with thechanges in the electrical property being indicative of double-strandedregions of the target analyte and of the flap regions. In FIGS. 10 a-10d, the first pair of electromotive electrodes 304, 304′ and the currentsource 306 have been omitted for clarity. In FIG. 10 a, the fluidicchannel 302 contains a target analyte 50 traveling therethrough. Anelectrical property, in this case electrical potential, is measured andrecorded across the detection volume 314 by the detector electrodes 312,312′ and the detector 316. The target analyte 50 is a DNA fragment uponwhich has been formed a flap 24 using the methods described previously.The DNA fragment and/or the flap may be coated with a binding moiety,such as the protein RecA, to enhance detection.

Prior to the entry of the target analyte 50 into the detection volume314, a substantially constant voltage 322 is measured across thedetection volume. This voltage is shown in the waveform 320 of FIG. 10a. As the target analyte 50 enters the detection volume 314, it causesan interruption or decrease in the electrical property measured in thedetection volume. This interruption or decrease causes a first trough324 to be exhibited in the waveform 320.

FIG. 10 b shows the device and waveform 320 once the portion of thetarget analyte 50 including the flap 24 has entered the detection volume314. Entry of the flap 24 into the detection volume 314 causes a furtherinterruption or decrease in the electrical property measured in thedetection volume. This further interruption or decrease causes a secondtrough 326 to be exhibited in the waveform 320.

In FIG. 10 c, the portion of the target analyte 50 containing the flap24 has exited the detection volume 314; however, a distal portion of thetarget analyte 50 may still be present in the detection volume. As aresult, the waveform 320 has returned to a level 328 approximating thatdetected when the initial portion of the analyte first entered thedetection volume.

Finally, as shown in FIG. 10 d, the target analyte 50 has fully exitedthe detection volume 314. As a result, the waveform 320 has returned toa level 330 approximating that detected prior to initial entry of theanalyte into the detection volume. Analysis of the waveform 320 permitsdifferentiation between double-stranded DNA and flap regions of thetarget analyte based, at least in part, on the detected changes in theelectrical property, to thereby determine nick locations and map atleast a portion of the double-stranded DNA template.

Another embodiment of a fluidic channel apparatus is shown in FIG. 11.In FIG. 11, the apparatus 400 comprises a fluidic micro-channel ornano-channel 402. As before, the fluidic channel may be a micro-channelhaving a width selected from a range of about 1 μm to about 25 μm or anano-channel having a width selected from a range of about 10 nm toabout 1 μm. In the case of a micro-channel, the depth may be selectedfrom a range of about 200 nm to about 5 μm, whereas in the case of anano-channel, the depth may be selected from a range of about 10 nm toabout 1 μm. In either case, the channel may have a length selected froma range of about 1 μm to about 10 cm.

A first pair of electromotive electrodes 304, 304′ is connected to avoltage source 306 and positioned in a spaced apart relationship in thechannel. When a potential is applied to the electromotive electrodes,these electrodes provide an electrical current along the channel and maybe used to provide or enhance a driving force 408 to an analyte 410 inthe channel. Other driving forces such as pressure or chemical gradientsare contemplated as well. Multiple detector electrodes 412, 414, 416,418, are positioned preferably perpendicular to the channel in a spacedapart relationship to define a plurality of detection volumes betweenadjacent detector electrodes. Thus, as seen in FIG. 11, detectorelectrodes 412 and 414 define detection volume 420, detector electrodes414 and 416 define detection volume 422, and detector electrodes 416 and418 define detection volume 424. The detector electrodes are eachconnected to detectors 426, 428, 430 such as voltmeters, which monitoran electrical property in each detection volume. In the embodiment wherethe detectors are voltmeters, a drop in electrical potential is measuredacross each detection volume. Operation of the apparatus is similar tothat of the system of FIG. 10, with the exception that additionalwaveforms are generated due to the presence of additional detectionvolumes. The additional waveforms may be combined to further improve thequality of the data being generated by the device.

It should be understood that number of detector electrodes and detectionvolumes is not intended to limited to those depicted in FIG. 11. Rather,any number of detection volumes may be included along the length of thefluidic channel. Further, the detector electrodes and detection volumesneed not be evenly spaced, evenly sized or directly adjacent to oneanother. Various detection volume sizes, spacing and configurations arecontemplated.

Both the nanopore apparatus and the fluidic channel apparatus allowdetection of an analyte as well as detection of a flap formed on thatanalyte. Furthermore, relative or absolute positional information of theflap may be obtained. Since, when using a known nicking endonuclease aspecific recognition sequence is known, determination of the location ofthe flap allows determination of the location of the known recognitionsequence. This in turn, allows the biomolecule to be mapped. Therepeated use of different nicking endonucleases allows greatercomplexity, i.e., multiple recognition sequences, to be combined andmapped.

EQUIVALENTS

Several of the illustrated examples show DNA templates having a singlenick formed thereon, with a single flap being formed thereafter.Embodiments of the present invention are not intended to be limited assuch; rather, it is contemplated that a nicking endonuclease may form aplurality of nicks at sequence specific locations on a first DNA strand.

Those skilled in the art will readily appreciate that all parameterslisted herein are meant to be exemplary and actual parameters dependupon the specific application for which the methods and materials ofembodiments of the present invention are used. It is, therefore, to beunderstood that the foregoing embodiments are presented by way ofexample only and that, within the scope of the appended claims andequivalents thereto, the invention may be practiced otherwise than asspecifically described. Thus, numerous variations and modifications willbe apparent to those skilled in the art. All such variations andmodifications are intended to be within the scope of the presentinvention as defined in the appended claims.

What is claimed is: 1.-12. (canceled)
 13. A target analyte comprising adouble-stranded DNA template having at least one single-stranded, flapregion extending therefrom, at least a portion of the double-strandedDNA template and the single-stranded flap region being coated with abinding moiety that enhances electrical detection of the double-strandedDNA template and the single-stranded flap region.
 14. The target analyteof claim 13, wherein the binding moiety comprises a protein.
 15. Thetarget analyte of claim 14, wherein said protein includes one or moreproteins selected from the group consisting of RecA, T4 gene 32 protein,f1 geneV protein, human replication protein A, Pf3 single-strandedbinding protein, adenovirus DNA binding protein, and E. colisingle-stranded binding protein.
 16. A method for preparing andanalyzing a target analyte, the method comprising: a. providing adouble-stranded DNA template having first and second DNA strands, eachstrand having a 5′ end and a 3′ end, b. contacting the template with anicking endonuclease to form nicks at sequence-specific locations on thefirst DNA strand, c. conducting a first base extension reaction on thefirst DNA strand along the corresponding region of the second DNAstrand, said reaction starting at each nick and progressing toward the3′ end of the first DNA strand to thereby form single-stranded flapregions on the double-stranded DNA template adjacent to thesequence-specific nicking locations, d. conducting a second baseextension reaction on at least one flap region to form at least onedouble-stranded flap, to thereby prepare the target analyte, e.translocating the target analyte through a fluidic channel, the fluidicchannel comprising at least a pair of detector electrodes defining adetection volume therein, and e. monitoring changes in an electricalproperty across a fluidic channel as the target analyte is translocatedtherethrough, the changes in the electrical property big indicative ofdouble-stranded regions of the target analyte and of the single-strandedflap regions.
 17. The method of claim 16, wherein the nickingendonuclease comprises one or more endonucleases selected from the groupconsisting of Nb.BbvCI, Nb.BsmI, NbBsrDI, Nb.BtsI, Nt.AlwI, Nt.BbvCI,Nt.BsmAI, Nt.BspQI, Nt.BstNBI, and Nt.CviPII.
 18. The method of claim16, wherein the first base extension reaction comprises contacting thefirst DNA strand with a polymerase, one or more nucleotides, a ligase,or any combination thereof.
 19. The method of claim 16, wherein thesecond base extension reaction comprises contacting at least one flapregion with a polymerase, one or more nucleotides, a ligase, or anycombination thereof.
 20. (canceled)
 21. A method for preparing andanalyzing a target analyte, the method comprising: a. providing adouble-stranded DNA template having first and second DNA strands, eachstrand having a 5′ end and a 3′ end, b. contacting the template with anicking endonuclease to form nicks at sequence-specific locations on thefirst DNA strand, c. conducting a first base extension reaction on thefirst DNA strand along the corresponding region of the second DNAstrand, said reaction starting at each nick and progressing toward the3′ end of the first DNA strand to thereby form single-stranded flapregions on the double-stranded DNA template adjacent to thesequence-specific nicking locations, and d. conducting a second baseextension reaction on at least one flap region to form at least onedouble-stranded flap, to thereby prepare the target analyte; e.translocating the target analyte through a nanopore while two electrodesproduce a measurable current parallel to movement of the target analyte;and f. monitoring changes in an electrical property across the nanoporeas the target analyte is translocated therethrough, the changes in theelectrical property being indicative of double-stranded regions of thetarget analyte and of the flap regions.
 22. The method of claim 21,further comprising: differentiating between double-stranded and flapregions of the target analyte based, at least in part, on the detectedchanges in the electrical property, to thereby determine nick locationsand map at least a portion of the double-stranded DNA template. 23.(canceled)
 24. The method of claim 16, wherein the fluidic channelcomprises a micro-channel or a nano-channel.
 25. (canceled)
 26. Themethod of claim 16, further comprising: differentiating betweendouble-stranded and flap regions of the target analyte based, at leastin part, on the detected changes in the electrical property, to therebydetermine nick locations and map at least a portion of thedouble-stranded DNA template.
 27. The method of claim 16, furthercomprising: coating the target analyte with a binding moiety.
 28. Thetarget analyte of claim 27, wherein the binding moiety comprises aprotein.
 29. The target analyte of claim 28, wherein said proteinincludes one or more proteins selected from the group consisting ofRecA, T4 gene 32 protein, f1 geneV protein, human replication protein A,Pf3 single-stranded binding protein, adenovirus DNA binding protein, andE. coli single-stranded binding protein. 30.-35. (canceled)
 36. Themethod of claim 16, further comprising: differentiating betweendouble-stranded and flap regions of the target analyte based, at leastin part, on the detected changes in the electrical property, to therebydetermine nick locations and map at least a portion of thedouble-stranded DNA template.
 37. A target analyte comprising adouble-stranded DNA template having one or more double-stranded flapregions extending therefrom, wherein at least a portion of thedouble-stranded template and one or more of the double-stranded flapregions are coated with a binding moiety that enhances electricaldetection of the template and the flaps.
 38. (canceled)
 39. The targetanalyte of claim 37, wherein the binding moiety comprises a protein. 40.The target analyte of claim 39, wherein said protein includes one ormore proteins selected from the group consisting of RecA, T4 gene 32protein, f1 geneV protein, human replication protein A, Pf3single-stranded binding protein, adenovirus DNA binding protein, and E.coli single-stranded binding protein. 41.-45. (canceled)
 46. A methodfor preparing and analyzing a target analyte, the method comprising: a.providing a double-stranded DNA template having a first and a second DNAstrand, each DNA strand having a 5′ end and a 3′ end, b. contacting thedouble-stranded DNA template with a nicking endonuclease to form a nickat a sequence-specific nicking location on the first DNA strand, c.conducting a base extension reaction on the first DNA strand along acorresponding region of the second DNA strand, said reaction starting atthe nick and progressing toward the 3′ end of the first DNA strand tothereby form a single-stranded flap on the double-stranded DNA templateadjacent to the sequence-specific nicking location, and d. coating thesingle-stranded flap with a binding moiety that selectively binds withsingle-stranded DNA to thereby prepare the target analyte; e.translocating the target analyte through a fluidic channel, the fluidicchannel comprising at least a pair of detector electrodes defining adetection volume therein; and f. monitoring changes in an electricalproperty across the fluidic channel as the target analyte istranslocated therethrough, the changes in the electrical property beingindicative of double-stranded regions of the target analyte and of thesingle-stranded flap regions.
 47. The method of claim 46, wherein thenicking endonuclease comprises one or more endonucleases selected fromthe group consisting of Nb.BbvCI, Nb.BsmI, NbBsrDI, Nb.BtsI, Nt.AlwI,Nt.BbvCI, Nt.BsmAI, Nt.BspQI, Nt.BstNBI, and Nt.CviPII.
 48. The methodof claim 46, wherein the base extension reaction comprises contactingthe first DNA strand with a polymerase, one or more nucleotides, aligase, or any combination thereof.
 49. The method of claim 46, whereinthe binding moiety comprises a protein.
 50. (canceled)
 51. A method ofclaim 50, further for preparing and analyzing a target analyte, themethod comprising: a. providing a double-stranded DNA template having afirst and a second DNA strand, each DNA strand having a 5′ end and a 3′end, b. contacting the double-stranded DNA template with a nickingendonuclease to form a nick at a sequence-specific nicking location onthe first DNA strand, c. conducting a base extension reaction on thefirst DNA strand along a corresponding region of the second DNA strand,said reaction starting at the nick and progressing toward the 3′ end ofthe first DNA strand to thereby form a single-stranded flap on thedouble-stranded DNA template adjacent to the sequence-specific nickinglocation, and d. coating the single-stranded flap with a binding moietythat selectively binds with single-stranded DNA, to thereby prepare thetarget analyte; e. translocating the target analyte through a nanopore,while two electrodes produce a measurable current parallel to movementof the target analyte; and f. monitoring changes in an electricalproperty across the nanopore as the target analyte is translocatedtherethrough, the changes in the electrical property being indicative ofdouble-stranded regions of the target analyte and of the single-strandedflap regions.
 52. The method of claim 51, further comprising:differentiating between double-stranded and single-stranded flap regionsof the target analyte based, at least in part, on the detected changesin the electrical property, to thereby determine nick locations and mapat least a portion of the double-stranded DNA template.
 53. (canceled)54. The method of claim 46, wherein the fluidic channel comprises amicro-channel or a nano-channel.
 55. (canceled)
 56. The method of claim46, further comprising: differentiating between double-stranded andsingle-stranded flap regions of the target analyte based, at least inpart, on the detected changes in the electrical property, to therebydetermine nick locations and map at least a portion of thedouble-stranded DNA template.
 57. A target analyte comprising adouble-stranded DNA template having at least one single-stranded, flapregion extending therefrom, the single-stranded flap region being coatedwith a binding moiety that enhances electrical detection thereof.
 58. Amethod for preparing and analyzing a target analyte, the methodcomprising: a. providing a double-stranded DNA template having first andsecond DNA strands, each strand having a 5′ end and a 3′ end, b.contacting the template with a nicking endonuclease to form nicks atsequence-specific locations on the first DNA strand, c. conducting afirst base extension reaction on the first DNA strand along thecorresponding region of the second DNA strand, said reaction starting ateach nick and progressing toward the 3′ end of the first DNA strand tothereby form single-stranded flap regions on the double-stranded DNAtemplate adjacent to the sequence-specific nicking locations, d.conducting a second base extension reaction on at least onesingle-stranded flap region to form at least one double-stranded flap,e. adding a single-stranded extension to the double-stranded flap, andf. hybridizing one or more probes to the single-stranded extension, tothereby prepare the target analyte, g. translocating the target analytethrough a fluidic channel, the fluidic channel comprising at least apair of detector electrodes defining a detection volume therein, and h.monitoring changes in an electrical property across the fluidic channelas the target analyte is translocated therethrough, the changes in theelectrical property being indicative of double-stranded regions of thetarget analyte and of the single-stranded flap regions.
 59. The methodof claim 58, wherein the single-stranded extension added to thedouble-stranded flap is a single-stranded polyT extension.
 60. Themethod of claim 59, wherein the probes comprise polyA oligomers.
 61. Themethod of claim 58, wherein the single-stranded extension added to thedouble-stranded flap is a single-stranded polyA extension.
 62. Themethod of claim 61, wherein the probes comprise polyT oligomers.
 63. Themethod of claim 58, wherein the probes are tagged.
 64. (canceled) 65.The method of claim 58, wherein the single-stranded extension is addedto the double-stranded flap using a terminal transferase.
 66. The methodof claim 58, wherein the single-stranded extension is at least 100 basesin length.
 67. A target analyte comprising a double-stranded DNAtemplate having one or more double-stranded flap regions extendingtherefrom, at least one double-stranded flap region having asingle-stranded extension added thereto.
 68. The target analyte of claim67 further including at least one probe hybridized to thesingle-stranded extension.
 69. The target analyte of claim 67, whereinthe single-stranded extension comprises a single-stranded polyTextension.
 70. The target analyte of claim 67, wherein thesingle-stranded extension comprises a single-stranded polyA extension.71. The target analyte of claim 68, wherein the probe comprises a polyAoligomer or a polyT oligomer.
 72. The target analyte of claim 68,wherein the probe is tagged.
 73. The target analyte of claim 72, whereinthe probe is tagged using a gold particle.
 74. (canceled)
 75. A methodfor preparing and analyzing a target analyte, the method comprising: a.providing a double-stranded DNA template having first and second DNAstrands, each strand having a 5′ end and a 3′ end, b. contacting thetemplate with a nicking endonuclease to form nicks at sequence-specificlocations on the first DNA strand, c. conducting a first base extensionreaction on the first DNA strand along the corresponding region of thesecond DNA strand, said reaction starting at each nick and progressingtoward the 3′ end of the first DNA strand to thereby formsingle-stranded flap regions on the double-stranded DNA templateadjacent to the sequence-specific nicking locations, d. conducting asecond base extension reaction on at least one single-stranded flapregion to form at least one double-stranded flap, e. adding asingle-stranded extension to the double-stranded flap, and f.hybridizing one or more probes to the single-stranded extension, tothereby prepare the target analyte, g. translocating the target analytethrough a nanopore, while two electrodes produce a measurable currentparallel to movement of the target analyte; and h. monitoring changes inan electrical property across the nanopore as the target analyte istranslocated therethrough, the changes in the electrical property beingindicative of double-stranded regions of the target analyte and of thesingle-stranded flap regions.
 76. The method of claim 75, furthercomprising: differentiating between double-stranded and single-strandedflap regions of the target analyte based, at least in part, on thedetected changes in the electrical property, to thereby determine nicklocations and map at least a portion of the double-stranded DNAtemplate.
 77. (canceled)
 78. The method of claim 58, wherein the fluidicchannel comprises a micro-channel or a nano-channel.
 79. (canceled) 80.The method of claim 58, further comprising: differentiating betweendouble-stranded and single-stranded flap regions of the target analytebased, at least in part, on the detected changes in the electricalproperty, to thereby determine nick locations and map at least a portionof the double-stranded DNA template.
 81. The method of claim 16, whereinthe electrical property is an electrical potential or a current.
 82. Themethod of claim 21, wherein the electrical property is an electricalpotential or a current.
 83. The method of claim 46, wherein theelectrical property is an electrical potential or a current.
 84. Themethod of claim 51, wherein the electrical property is an electricalpotential or a current.
 85. The method of claim 58, wherein theelectrical property is an electrical potential or a current.
 86. Themethod of claim 75, wherein the electrical property is an electricalpotential or a current.